Cancer Cell Biology
High expression levels of collagenase-1 and stromelysin-1 correlate with shorter disease-free survival in human metastatic melanoma
Article first published online: 22 OCT 2001
Copyright © 2002 Wiley-Liss, Inc.
International Journal of Cancer
Volume 97, Issue 4, pages 432–438, 1 February 2002
How to Cite
Nikkola, J., Vihinen, P., Vlaykova, T., Hahka-Kemppinen, M., Kähäri, V.-M. and Pyrhönen, S. (2002), High expression levels of collagenase-1 and stromelysin-1 correlate with shorter disease-free survival in human metastatic melanoma. Int. J. Cancer, 97: 432–438. doi: 10.1002/ijc.1636
- Issue published online: 29 DEC 2001
- Article first published online: 22 OCT 2001
- Manuscript Accepted: 30 JUL 2001
- Manuscript Revised: 25 JUN 2001
- Manuscript Received: 9 MAR 2001
- Turku University Central Hospital
- Blida Maunula Foundation of the University of Turku
- Cancer Research Foundation of Finland
- Sigrid Juselius Foundation
- Finnish Cultural Foundation
- Academy of Finland
- metastatic melanoma;
- disease-free survival
Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of degrading extracellular matrix. Their role has been emphasized in tumor invasion, metastasis and tumor-induced angiogenesis. We studied the expression of collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) in 70 melanoma metastases obtained from 56 patients treated with combined chemoimmunotherapy. The patients were divided into 2 groups using a cut-off point of 0% for MMP-1 expression and 20% for MMP-3 expression. We found that patients with MMP-1 positive metastases (n = 38) had significantly shorter disease-free survival compared to patients with MMP-1 negative metastases (n = 18) (median 11.2 vs. 17.0 months, p = 0.0383). The disease-free survival of patients with high levels of MMP-3 expression in their metastases (≥20% positive tumor cells, n = 14) was also significantly shorter compared to patients with lower levels of expression (n = 42) (median 5.1 vs. 14.0 months, p = 0.0294). The expression of MMP-13 did not correlate to survival parameters. We also found that the presence of melanin, a pigment produced by melanocytes, correlated with high expression levels of MMP-1 (p = 0.0002), MMP-3 (p < 0.0001) and MMP-13 (p = 0.0009). The high expression levels of MMP-13 were also associated with the presence of visceral metastases (p = 0.0284). Our findings suggest that MMP-1 and -3 may have a special role in melanoma metastasis formation and thus they could be used to measure the biological activity of the disease. © 2001 Wiley-Liss, Inc.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases that can degrade essentially all extracellular matrix components. At present, the human MMP gene family contains 21 members that can be divided into subgroups of collagenases, gelatinases, stromelysins, membrane-type MMPs (MT-MMPs) and novel MMPs according to their substrate specificity and primary structure.1, 2 There are specific tissue inhibitors of metalloproteinases (TIMPs) that can inhibit the activity of MMPs. The balance between MMPs and their inhibitors is essential in many physiological conditions as well as in pathological conditions such as rheumatoid arthritis, atherosclerosis, tumor invasion, metastasis and tumor-induced neovascularization.3, 4
Members of the collagenase subgroup, i.e., collagenase-1 (MMP-1), collagenase-2 (MMP-8) and collagenase-3 (MMP-13), are only neutral proteinases capable of degrading native fibrillar collagens of types I, II, III and V. After initial cleavage by collagenases followed by denaturation the fibrillar collagens are further degraded by gelatinases. MMP-13 has exceptionally wide substrate specificity compared to other MMPs. In addition to native fibrillar collagens it degrades type IV, X and XIV collagens, tenascin, fibronectin and the aggrecan core protein.2 Stromelysin-1 (MMP-3) was one of the first proteinases found to be associated with cancer.5 MMP-3 can degrade fibronectin, type IV, V, IX and X collagens, elastin, laminins, gelatin and proteoglycan core proteins and furthermore it also plays a role in activation of other proMMPs, including MMP-1 and MMP-13.1
MMP-1 is expressed by various types of cells in culture and in vivo and its expression has been associated with chronic cutaneous wounds as well as chronic intestinal ulcers.6 High level of MMP-1 expression correlates with poor prognosis in many types of cancer, i.e., in colorectal cancer,7 esophageal cancer8 and gastric cancer9 as well as in pancreatic carcinoma. 10 In addition, abundant expression of MMP-1 correlates with tumor invasion and nodal involvement in primary oral squamous cell carcinoma specimen.11
MMP-3 is expressed by epithelial cells, such as keratinocytes and fibroblasts and by various types of malignant cells.1 Its expression is related to lymph node metastasis and vascular invasion in squamocellular carcinomas of the esophagus.12 MMP-3 may be able to activate proMMP-9 in colorectal carcinoma resulting in cancer progression and metastasis.13 MMP-3 is synthesized both by tumor and peritumoral stromal cells in human invasive breast carcinomas but its expression levels are approximately the same as in normal breast epithelia.14 In mice, MMP-3 can act as a natural mammary tumor promoter.15
The physiological expression of MMP-13 is limited to situations in which rapid and effective remodeling of collagenous matrix is required, i.e., fetal bone development and postnatal bone remodeling16, 17 and gingival wound repair.18 The expression of MMP-13 associates with excessive breakdown of collagenous matrix in osteoarthritic cartilage,19 rheumatoid synovium,17 chronic cutaneous ulcers, intestinal ulcerations20 and periodontitis.21 MMP-13 is also expressed in malignant tumors including breast carcinomas,22 squamous cell carcinomas of the head and neck23 and the vulva,24 chondrosarcomas,25 basal cell carcinomas of the skin26 and urinary bladder carcinomas.27
Melanoma arises from melanocytes, cells that in normal skin are located in the basal layer of epidermis. These melanocytes produce a pigment called melanin, that can protect skin from UV-radiation. In human malignant melanoma patient particular melanin metabolites in urine or serum can predict the dissemination of the disease.28 Similarly, the increased expression levels of gelatinases (MMP-2 and -9) or collagenases (MMP-1, -8 and -13) have been shown to correlate with melanoma tumor invasiveness29, 30 or progression.31 High expression level of MMP-2 can predict poor outcome in uveal melanoma32 and de novo expression of MMP-9 in neoplastic melanocytes early invasion in melanoma of skin.33 Recently, Hofmann et al.34 have shown that the coexpression of MT1-MMP, TIMP-2 and MMP-2 correlates with tumor progression both in a xenograft model and in human cutaneous lesions of primary and metastatic melanoma. In other mouse models the overexpression of TIMP-2 has been associated with reduced tumor growth35 or melanoma cell invasion36 as well as inhibition of lung metastasis formation.37
In our study we have analyzed the expression of MMP-1, MMP-3 and MMP-13, in the metastases of 56 melanoma patients treated with combined chemoimmunotherapy. We show that the disease-free survival of the patients with positive tumor cells for MMP-1 or MMP-3 (≥20%) is significantly shorter than that of the patients with negative or lower (<20%) expression levels of the same MMPs, respectively. Similarly, there is a tendency toward shorter disease-free survival among patients with metastasis expressing MMP-13. In addition, the increased expression levels of all the studied MMPs correlate with the presence of melanin in the tumor samples.
MATERIAL AND METHODS
Patients and tissue samples
The analyzed patient group consisted of 56 patients with metastatic melanoma. These patients had been treated according to a phase II trial in the Department of Oncology of Helsinki University Central Hospital where they received a 4-drug chemotherapy composed of dacarbazine, vincristine, bleomycin and lomustine (DOBC) combined with natural leukocyte interferon-α.38, 39 The patients recruited in these studies had signs of progressive, inoperable, histologically verified metastatic melanoma. In some patients metastases had been previously operated and they were attending to our study when they progressed. In other patients the metastatic disease was only biopsy-proven. 70% of the patients had no previous treatment, 9% had previous chemotherapy, 16% had previous radiotherapy and 5% had previous therapy with interferon-α or interleukin-2. There were 26 patients who had no visceral metastases. Twenty-three patients had either skin or lymph node metastasis and 3 had both. Twenty-nine patients had visceral metastases, 17 had either skin or lymph node metastasis combined with visceral metastases and 12 had metastases in multiple organs. Complete lymph node dissections were not performed. Before treatment, a full medical examination was carried out. Investigations included a chest X-ray, abdominal ultrasound or computerized topography scan, a bone scan and blood biochemistry. The estimation of tumor load of a patient and the number of organs affected with metastases is based on these mentioned studies. Because of serious side effects 1 patient was withdrawn from the therapy. In the beginning of the chemoimmunotherapy the performance status of the patients was evaluated. Patient characteristics are summarized in Table I.
|Median (range)||47 (24–74)|
|Disease-free survival (months)|
|Median (range)||13 (0.4–200)|
|Overall survival (months)|
|Median (range)||39 (5–259)|
|Survival after the initiation of chemoimmunotherapy (months)|
|Median (range)||10 (0.9–104)|
|Treated patients2 (n)||55|
|No previous treatment (n)||38|
|Performance status3 (n)|
|Presence of visceral metastases3 (n)|
|Number of organs with metastases3 (n)|
|Three of more organs||12|
|Treatment response5 (n)|
Altogether 70 samples were collected for the analysis before and during the treatment of metastatic melanoma between the years of 1989 and 1995. Twenty-eight of the samples were skin or subcutaneous metastases, 36 lymph node metastases, 2 brain and 2 soft tissue metastases and 1 metastasis from both parotis and orbit, respectively. Fifty-two of the samples were obtained before treatment and eighteen of the samples were obtained during the ongoing treatment. Characteristics of the tumor specimens are summarized in Table II. There were 10 patients with more than 1 tumor sample. There were 3 patients with both pre- and on-treatment samples, 1 patient with 2 pre- and 2 on-treatment samples, 2 patients with 2 separate pre-treatment samples and 1 on-treatment sample, 3 patients with 2 different pre-treatment samples and 1 patient with 2 different on-treatment samples.
|Sites of metastases analysed|
|On-treatment (progression) sample||18|
|Expression of MMP-1|
|Expression of MMP-3|
|Expression of MMP-13|
After surgical excision, the tumor samples were fixed in phosphate-buffered formalin (pH 7.2), dehydrated in a graded alcohol series and xylene and embedded in paraffin, according to our normal routine procedure. All tumor specimens were re-examined by specialist pathologists and the diagnosis of melanoma was confirmed. The tissue specimens were cut to 6 μm sections for immunohistochemical stainings.
In the analysis for disease-free survival only the first metastasis sample from each patient was studied.
To detect the expression of MMP-1 and -13 monoclonal antibodies anti-MMP-1 (IM35L) and anti-MMP-13 (IM44L; both from Oncogene Research Products, Cambridge, MA) were used. For detection of MMP-3 a polyclonal anti-MMP-3 antibody (AB810; Chemicon International Inc., Temecula, CA) was used. Rabbit anti-cow anti-S100 antibody (20311, DAKO A/S, Glostrup, Denmark) was used to detect malignant melanoma cells in studied tumors. The dilutions of 1:500 for MMP-1 and -3, 1:100 for MMP-13 and 1:1,000 for S100 in 0.3% BSA in PBS were used. Mouse monoclonal antibody for platelet endothelial cell adhesion molecule-1 (1:50, PECAM-1, CD31) was used for assessing vascularity (DAKO A/S, Glostrup, Denmark). The DAKO StreptABComplex/HRP Duet Mouse/Rabbit Kit was employed for immunoperoxidase staining (DAKO A/S, Glostrup, Denmark).
The melanoma metastases were stained with S100 antibody, which specifically recognizes melanoma cells. The majority of melanoma cells were S100 positive. S100 stainings were performed to show that in these tumors only melanoma cells express the studied MMPs and to separate melanoma cells from lymphocytes in lymph node metastases. The immunohistochemical staining procedure was optimized in preliminary experiments by using 6 μm paraffin sections of healthy adult human skin and breast cancer tissue. In the skin the expression of MMP-1 was limited to areas in which rapid remodeling occurs, i.e., hair follicles and in breast tissue to milk ducts. Epithelial cells expressed MMP-3 and breast cancer cells MMP-13. The same staining conditions and antibody concentrations were then used to screen the expression of metalloproteinases in the melanoma metastasis specimen. The final conditions used were as follows: The formalin-fixed, paraffin-embedded sections were first dehydrated and then gently cooked in citrate buffer for 15 min using a microwave oven. After cooling in room temperature and washing with phosphate-buffered saline (PBS), a quenching of endogenous peroxidase with 30% hydrogen peroxide in methanol for 30 min was performed. After washing in PBS, non-specific binding was blocked by incubation with normal goat serum diluted in 0.3% BSA in PBS for 30 min. The primary antibodies were applied on the slides and incubated at room temperature for one hour. After a wash in PBS, the secondary biotinylated antibody (DAKO Kit) was added and incubated at room temperature for 30 min. Slides were washed with PBS and avidin-biotin-peroxidase complex (working solution DAKO Kit) was applied and incubated at room temperature for 30 min. Slides were washed again and peroxidase activity was developed in a 3-amino-9-ethylcarbazole (AEC; Sigma, St. Louis, MO) solution with hydrogen peroxide. Sections were counterstained with Mayer's haematoxylin.
In addition, sections of the 17 samples containing melanin were dehydrated, cooked in citrate buffer, washed in PBS and counterstained with Mayer's haematoxylin (Fig. 1a). To exclude the possibility that melanin could non-specifically have bound secondary and tertiary antibodies, a control sample was stained using the exact same protocol as described above with the exemption of not using the primary antibody (Fig. 1b).
Evaluation of MMP expression
The expression of the MMPs was assessed without knowing the clinical data. The slides were first screened to get an overview of the general staining pattern. By using a light microscope connected to a camera and a computer program AnalySIS, v 3.00 (Soft Imaging System GmbH, Münster, Germany) 4 pictures of each slide were taken using 4 × magnification. The randomly taken pictures covered most of the tumor area. The pictures were analyzed using an Imaging Research M4-computer program (v 3.0, Imaging Research Inc., St. Catharines, Ontario, Canada). This program analyses the red color in the positively stained cells and counts the area of those cells in pixels. This same program also counted the total tumor area in pixels. The percentage of the positively stained tumor cells from the whole tumor area was counted and used for further analysis.
Evaluation of melanin expression
There were 17 samples containing melanin, a red-brown pigment produced by melanocytes. This pigment in the cells appeared to be problematic because the computer program was not able to separate melanin and positive staining for a MMP. Therefore the 17 samples expressing melanin were assessed differently, by two ways. Melanin expressing tumors were stained only with haematoxylin-counterstaining as described in Material and Methods. These sections served as controls when melanin was separated from MMP staining in exactly the same tumor area. Then the Imaging Research M4-computer program was used to calculate the area of melanin from the control sample. The same intensity and color settings were used to assess the area stained for MMPs and the area of melanin was reduced from these results. Secondly, the amount of positively stained cells was estimated (by J.N.) using a light microscope. The final results were based on an agreement between these 2 ways of assessing the samples.
Evaluation of vascularity and proliferation
We had some data available from previous experiments. Thus, it was possible to correlate the expression of the studied MMPs to vascularization or MIB-1 expression levels in the same metastases. There were 65 of the samples available for simultaneous study for vascularity and MMP expression and 61 samples available for study for MIB-1 and MMP expression.
Blood vessel quantification has been previously described in detail.40 Shortly, tumor areas with highest vascular density were first identified under light microscopy at 40× magnification and then the signals from 3–6 single fields at 250× magnification were counted. Every separate group of clearly positive endothelial cells were considered as a blood vessel. Vessel count was expressed as the mean number of vessels in these areas.
The results from immunohistochemistry and clinical data were analyzed with the StatView™ package for Windows, v 4.53 (Abacus Concepts Inc., Berkeley, CA). The Mann-Whitney U-test was used in assessing the correlation between MMP-expression and pigmentation. Cumulative survival curves for disease-free survival was drawn by the Kaplan-Meier method and the difference between the curves was analyzed by the Mantel-Cox (logrank) test.
Expression of MMP-1, MMP-3 and MMP-13 in melanoma metastases
The percentage of positively stained tumor cells of the total tumor area was counted as an average of four separate, randomly chosen microscope fields by using Imaging Research M4-computer program. The percentages of positively stained cells varied from 0–93.5% in the samples stained for MMP-1, with a median value of 0.3% and a mean value of 11.8% (Fig. 1a,b). In the samples stained for MMP-3 the percentages varied from 0–100% with a median of 4.0% and a mean value of 20.5% (Fig. 1c,d) In MMP-13 samples percentages varied from 0–85.3% with a median of 10.1% and a mean value of 20.2% (Fig. 1e,f) (Table II). The lymph node metastases were also stained with a melanocyte specific anti-S100 antibody to show the presence of metastatic tumor cells (Fig. 1g–i).
Expression of MMPs and pigmentation
Seventeen of the samples contained melanin and the rest of the samples (n = 53) were non-pigmented (Table II). Melanin pigment was found in the samples, but it could be clearly separated from MMP stainings by different staining protocols. In Figure 2a a skin metastasis sample is stained only by haematoxylin counterstaining. In Figure 2b the same sample is stained by immunoperoxidase protocol but without a specific anti-MMP antibody. In Figure 2c–e the same sample is stained by immunoperoxidase protocol by using a specific antibody for MMP-1, MMP-3 or MMP-13, respectively. We found that the expression levels of the studied MMPs were higher in the pigmented samples than in non-pigmented ones. The pigmentation correlated with a higher MMP-1 value (mean 29.9%) than absent pigmentation (mean 5.9%) (p = 0.0002, Mann-Whitney U-test, Table II, Fig. 2c). Similar correlation were found also for MMP-3 and MMP-13: The mean expression levels of MMP-3 were 42.5% in pigmented samples and 13.5% in non-pigmented samples (p<0.0001, Table II, Fig. 2d). For MMP-13 the mean expression levels were 37.1% in pigmented samples and 14.8% in non-pigmented samples (p<0.0009, Table II, Fig. 2e).
Expression of MMPs and disease-free survival
The most appropriate cut-off points for dividing the results of the expression of MMPs into two groups were defined by giving different values for the cut-off point and determining the corresponding p-values using the Mantel-Cox (logrank) test to analyze Kaplan-Meier survival curves. This procedure of determining the cut-off point instead of using mean or median values has been described previously.41 The cut-off point that corresponded to the lowest p-value was used in the analysis. In this way the cut-off value was <0% for the samples stained for MMP-1 (Fig. 3a), ≥20% for the samples stained for MMP-3 (Fig. 3b) and ≥7% for the samples stained for MMP-13 (Fig. 3c).
Of the studied 56 patients 18 had metastases with the expression of MMP-1 below the cut-off point and 38 with the expression above the cut-off point (Fig. 1a,b). The same patient numbers for MMP-3 were 42 (below) and 14 (above) (Fig. 1c,d) and 29 (below) and 27 (above) for MMP-13 (Fig. 1e,f).
We found that those patients with no MMP-1 expression in their metastases (n = 18), had significantly longer disease-free survival when compared to those patients with metastases positive for MMP-1 (n = 38) (median 17.0 vs. 11.2 months, p = 0.0383, Logrank test, Fig. 4a). Similarly, patients with metastases with less than 20% of MMP-3 positive tumor cells (n = 42) had significantly longer disease-free survival than those with metastases with 20% or more positive tumor cells (n = 14) (median 14.0 vs. 5.1 months, p = 0.0294, Logrank test, Fig. 4b). Also, a tendency toward longer disease-free survival of those patients (n = 29) with less than 7% of MMP-13 expressing tumor cells was seen, though the difference to those patients (n = 27) with 7% or more MMP-13 expressing tumor cells was not statistically significant (median 16.7 vs. 10.7 months, p = 0.1365, Logrank test, Fig. 4c).
MMP expression levels and tumor vascularity or proliferation index
We had data available from the previous experiments and thus were able to study the possible relationship between MMP expression and tumor vascularity or proliferation index (MIB-1) in 65 or 61 sections of the same samples, respectively. 65 of the samples were simultaneously studied for vascularity and MMP expression. PECAM-1 (CD31) was used for assessing vascularity by immunohistochemical staining. Blood vessels were counted and the values for vascularity were given in blood vessel number/high power field. In these 65 samples the mean value for vascularity was 31 blood vessels per field and the results varied from 4–131 blood vessels per field. There was no correlation between the expression of MMP-1 (p = 0.1302), MMP-3 (p = 0.5585) or MMP-13 (p = 0.2512) and vascularity (Spearman rank).
Tumor proliferative activity was studied by MIB-1, a monoclonal antibody to a Ki-67 epitope, which can recognize all proliferating cells. Sixty-one of the samples were studied for both MIB-1 and MMP expression. The mean value for MIB-1 was 42% and the values varied from 9.5% to 80%. There was no statistical significance between the high expression levels of different MMPs and tumor MIB-1 value (MMP-1; p = 0.3700, MMP-3; p = 0.7752, MMP-13; p = 0.2332, Spearman rank).
Expression of MMPs and presence of visceral metastases
In literature the involvement of visceral organs with metastasis has been connected to poor prognosis. Tumor load has a major effect on the overall as well as to disease-free survival of the metastatic melanoma patients. We estimated the tumor load of a patient by measuring the amount of metastatic sites by a full pretreatment medical examination (see Material and Methods). There were 32 patients (57%) with metastases in 2 or more organs. Their metastatic disease was biopsy-proven, but all metastases were not surgically removed. We studied if the expression of MMPs did have any correlation to the number of metastatic sites or the presence of visceral metastases in the beginning of the chemoimmunotherapy. Patients with visceral metastases (n = 29) had significantly higher expression levels of MMP-13 than patients without visceral metastases (n = 26) (22% vs. 13%, p = 0.0284, Mann-Whitney U-test). No such association was found for MMP-1 (p = 0.7457) or MMP-3 (p = 0.3671) (Mann-Whitney U-test).
No association was found for the number of organs involved with metastases (1 organ, 2 organs, ≥3 organs, Table I) and the expression of MMP-1 (p = 0.8015), MMP-3 (p = 0.9601) or MMP-13 (p = 0.2583) (Kruskal-Wallis test).
Matrix metalloproteinases are a family of endopeptidases, which are usually first secreted and then, activated to degrade extracellular matrix proteins. Their expression is rapidly upregulated in situations where remodeling of matrix is needed e.g., in tumor cell invasion. MMPs are expressed in many types of human cancer both in vitro and in vivo.42 There is some recent work about the usefulness of MMPs as markers for patient survival in cancer. High expression levels of MMP-2, MMP-9, MT1-MMP and TIMP-2 in peritumoral stromal cells of ovarian carcinoma patients correlate with poor survival.43 Increased expression levels of MMP-2 have been associated with unfavorable outcome also in gastric cancer,44 in urothelial cancer45 and in early stage non-small cell lung cancer.46 Overexpression of MMP-9 is related to well differentiated hepatocellular carcinoma47 and to recurrence in early non-small cell lung carcinoma.48 MT1-MMP expression in tumor tissue is associated with invasiveness and poor prognosis of laryngeal carcinoma.49
In human malignant melanoma the increased expression levels of MMP-2 and MMP-9 have been shown to correlate with melanoma invasiveness29, 30 and progression.31 Elevated expression levels of MMP-1 and MMP-13 have also been connected to invasion of primary melanoma.30 Decreased expression of TIMPs has been documented in several studies of melanoma progression. Airola et al.31 have found, however, that the expression of both MMP-1 and MMP–13 as well as their inhibitors, TIMP-1 and TIMP–3, is associated with progression of primary cutaneous melanoma. In our previous work we have shown that high expression levels of MMP-1 or low expression levels of MMP-13 in the first melanoma metastases are associated with favorable treatment response.50 When we examined progression samples after chemoimmunotherapy, however, we could not find any correlation between treatment response and MMP expression suggesting that the presence of particular MMPs in melanoma metastases can predict tumor aggressiveness and thus effectiveness of a chosen therapy.50
In our study MMP levels were detected by immunohistochemical staining without measuring any protease activities. As the expression of most MMPs is low or undetectable in normal tissues, however, upregulation of their expression in malignant tumors in vivo has been taken to indicate that they are activated in tumor tissue. We found the expression of MMP-1 and high expression levels of MMP-3 to predict shorter disease-free survival of the patients. Tumor cells with low expression levels of MMP-1 or MMP-3 may need to use other strategies for metastatic spread than collagenolysis. Our work shows that the expression of MMP-1 or MMP-3 in a metastasis specimen might be used to measure disease activity and their presence may also suggest a need for a new treatment strategy. Nowadays synthetic matrix metalloproteinase inhibitors (MMPIs) are under development and already in different stages of clinical trials.51 In addition to tumor invasion many MMPIs can inhibit angiogenesis.52 Blood vessel density has been shown to be a prognostic predictor in patients with many types of cancer including primary malignant melanoma53, 54 and metastatic melanoma.40 In our results the expression levels of MMPs did not have any correlation to tumor vascularity. The results of the present study, however, suggest that MMPIs specifically targeted against MMP-1 and MMP-3 may be effective in inhibiting melanoma growth and metastasis by other mechanisms.
Melanin precursors and metabolites in blood have been associated with progression of melanoma.55, 56 Also, the melanin precursors 5-S-cysteinyldopa and the ratio of L-dopa to L-tyrosine in blood have been suggested to be the most specific and sensitive enough antigens associated with melanoma.56 Accumulation of melanin in melanocytes, however, has also been associated with senescence, better tolerance to UV irradiation and decrease of cell proliferation. This phenomenon is thought to be mediated by tumor suppresser genes p16INK, retinoblastoma protein and cyclin E.57 In other work with human melanoma cells, melanogenesis has been shown to be mediated by phosphatidylinositol 3-kinase (P13K)-Akt pathway.58 At least in human fibroblasts the expression of MMP-1 or MMP-3 can be regulated by the mitogen-activated protein kinases (MAPKs).59 MAPK pathways are arranged in cascades of three protein kinases, which are activated by phosphorylation in response to different stimuli, i.e., growth factors, cytokines or cellular stress.60 UV-radiation leads to cellular stress, which can activate both, c-Jun N-terminal kinase (JNK) and p38 dependent MAPK pathways. JNK is known to be necessary for apoptosis in response to ultra violet and gamma irradiation.61 Cross talk between P13K-dependent and MAPK signaling cascades occurs through specific activating proteins, i.e., B-Raf.62 In our study we found the presence of melanin in metastatic samples to correlate with the high expression levels of all the studied MMPs, MMP-1, MMP-3 and MMP-13. We suggest that the synthesis of these MMPs and melanin can be upregulated by similar, perhaps UV-irradiation induced mechanisms. Although no correlation between melanin expression and patient survival was detected, the possible relationship between MMP or melanin expression and tumor cell apoptosis should be further studied to understand the biology of melanoma metastasis formation.